Chimeric vaccine against tick-borne encephalitis virus

ABSTRACT

A live, attenuated chimeric virus vaccine against tick-borne encephalitis virus comprising the preM and E structural genes of the tick-borne encephalitis Langat virus and the non-structural genes of the mosquito-borne dengue virus. The live chimeric vaccine was administered intraperitoneally and exhibited complete attenuation in mice while at the same time providing protection against subsequent challenge with the virulent parental Langat virus.

RELATED APPLICATIONS

This application is a continuation and claims the benefit of priority ofU.S. patent application Ser. No. 09/518,036 filed Mar. 3, 2000, now U.S.Pat. No. 6,497,884, which is a continuation and claims the benefit ofpriority of International Pat. Appl. No. PCT/US98/21308 havinginternational filing date of Oct. 8, 1998, designating the United Statesof America and published in English, which claims the benefit ofpriority of U.S. Prov. Appl. No. 60/061,441, filed Oct. 8, 1997, all ofwhich are hereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to a chimeric virus vaccine againsttick-borne encephalitis virus (TBEV) and its other virulent relatives.More specifically, the invention relates to a chimeric virus comprisingthe Langat (LGT) virus preM and E structural protein genes linked to thenon-structural protein genes of a mosquito-borne flavivirus.

BACKGROUND OF THE INVENTION

The Flaviviridae family encompasses more than sixty antigenicallyrelated, positive strand RNA viruses within the arthropod-borneflavivirus genus, many of which are important human pathogens (Monath etal., Flaviviruses, in Virology, B. N. Fields et al., Eds., Raven Press,New York, pp. 961-1035, 1996). These include the mosquito-borne yellowfever virus, Japanese encephalitis virus, dengue viruses (DEN) and thetick-borne encephalitis viruses (TBEV), the latter being endemic in mostEuropean countries, Russia, India and North China. TBEV is transmittedexclusively by ticks and can be divided into two serologicallydistinguishable subtypes: the Eastern subtype (prototype strain Sofjin),prevalent in Siberian and Far Eastern regions of Russia, and the Westernsubtype (prototype strain Neudorfl), common in eastern and centralEurope. TBEV causes a serious encephalitic illness with a mortality rateranging from 1 to 30%.

Flaviviruses share the same genome organization:5′-C-preM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3′ in which the first threegenes code the capsid (C), premembrane (preM) and envelope (E) proteins,while the remainder of the genes encode nonstructural proteins. Homologybetween mosquito-borne and tick-borne flaviviruses is relatively low(Chambers et al., Annu. Rev. Microbiol. 44:649-688, 1990; Pletnev etal., Virology 174:250-263, 1990). However, homology among mosquito-borneflaviviruses or among tick-borne flaviviruses is relatively high(Iacoco-Connors et al., Virology 188:875-880, 1992).

Four serotypes of dengue virus are known (type 1 to type 4) which aredistinguishable by plaque reduction neutralization usingserotype-specific monoclonal antibodies and by less specific tests usingpolyclonal sera (Bancroft et al., Pan Am. Hlth. Org. Sci. Publ.375:175-178, 1979; Henchal et al., Am. J. Trop. Med. Hyg. 31:548-555,1982). The four dengue serotypes share a common genome organization. Thecomplete nucleotide sequences have been determined for dengue virustypes 3 and 4 and several strains of type 2 virus including themouse-neurovirulent New Guinea C mutant (Mackow et al., Virology159:217-228, 1987; Zhao et al., Virology 155:77-88, 1986; Osatomi etal., Virology 176:643-647, 1990; Irie et al., Gene 75:197-211, 1989;Mason et al., Virology 161:262-267, 1987; Hahn et al., Virology162:167-180, 1988).

Despite the considerable evolutionary distance between DEN and TBEV, aviable chimeric flavivirus was constructed which contained the C-preM-Eor preM-E structural protein genes of a virulent Far Eastern RussianTBEV with the remaining nonstructural protein genes and 5′- and3′-noncoding sequences derived from DEN4 [TBEV(CME)/DEN4 andTBEV(ME)/DEN4, respectively] (Pletnev et al., Proc. Natl. Acad. Sci.U.S.A. 89:10532-10536, 1992).

TBEV(ME)/DEN4 retained the neurovirulence in mice of its TBEV parentfrom which its preM and E genes were derived, but it lacked theperipheral neurovirulence of TBEV, i.e. the ability to spread from aperipheral site to the central nervous system (CNS) and cause fatalencephalitis. However, mice previously inoculated with the chimericvirus by a peripheral route were completely resistant to subsequentintraperitoneal challenge with a lethal dose of the highly virulentTBEV. Neurovirulence of this chimera was significantly reduced by asingle mutation introduced into its preM, E or nonstructural protein 1(NS1) viral protein (Pletnev et al., J. Virol. 67:4956-4963, 1993).These amino acid substitutions also caused a restriction in viralreplication in tissue cultures of both simian and mosquito cells.Nonetheless, parenteral inoculation of these further attenuated chimericmutants induced complete resistance in mice to fatal encephalitis causedby intracerebral inoculation of the neurovirulent TBEV(ME)/DEN4 chimera.

Langat (LGT) virus is the least virulent of all TBEV-complexflaviviruses, but is closely related antigenically to the highlyvirulent Far Eastern TBEV (Calisher et al., J. Gen. Virol. 70:37-43,1989; DeMadrid et al., J. Gen. Virol. 23:91-96, 1974; Iacoco-Connors etal., Virus Res. 43:125-136, 1996) and has a high level of sequencehomology thereto (Iacoco-Connors et al., Virology 188:875-880, 1992;Mandl et al., Virology 185:891-895, 1991; Shamanin et al., J. Gen.Virol. 71:1505-1515, 1990). LGT virus was tested as an experimental livevaccine against TBEV during the early 1970s (Ilenko et al., Bull. Wld.Hlth. Org. 39:425-431, 1968; Mayer et al., Acta. Virol. 19:229-236,1975; Price et al., Bull. Wld. Hlth. Org. 42:89-94, 1970). Several LGTstrains which were attenuated for mice and monkeys were isolated andtested in 800,000 adults; however, clinical trials were discontinuedwhen vaccination with one of the most attenuated vaccine candidates,Yelantsev virus, was associated with a very low frequency ofencephalitis, i.e. one case per 20,000 vaccinations (Mandl et al.,supra.)

Currently, an experimental TBEV vaccine produced by formalininactivation of TBEV is available; however, this vaccine has severallimitations. For example, the vaccine is not strongly immunogenic,therefore repeated vaccinations are required to generate a protectiveimmune response. Even when antibody responses to the vaccine arepresent, the vaccine fails to provide protective responses to the virusin 20% of the population. Therefore, there remains the need for a safe,more effective vaccine against TBEV. The present invention provides sucha vaccine.

SUMMARY OF THE INVENTION

One embodiment of the present invention is a viable chimeric recombinantflavivirus, comprising a first region of nucleic acid operably encodingpreM and E structural proteins of Langat virus operably linked to asecond region of nucleic acid operably encoding non-structural proteinsof a mosquito-borne flavivirus. Preferably, the Langat virus is theLangat wild type virus strain TP21 or its further attenuated mutant,Langat strain E5. Advantageously, the mosquito-borne flavivirus is adengue virus. In one aspect of this preferred embodiment, the denguevirus is type 4. Alternatively, the mosquito-borne flavivirus is yellowfever virus. According to another aspect of this preferred embodiment,the first region of nucleic acid also operably encodes capsid proteinfrom the mosquito-borne flavivirus or from Langat virus. In yet anotheraspect of this preferred embodiment, the recombinant flavivirus furthercomprising at least one mutation. Preferably, the recombinant flavivirusis incorporated within an expression vector. Advantageously, theexpression vector is a plasmid.

The present invention also provides a host cell stably transformed withthe recombinant flavivirus described above in a manner allowingexpression of said DNA construct. Preferably, the host cell isprokaryotic. In another aspect of this preferred embodiment, thetick-borne encephalitis virus is selected from the group consisting ofthe Eastern, Western, Omsk hemorrhagic fever, louping ill, Kyasanurforest disease, Negishi, or Powassan viruses.

Another embodiment of the present invention is a vaccine againsttick-borne encephalitis virus, comprising the chimeric recombinantflavivirus described above in a pharmaceutically acceptable carrier.Another embodiment is the vaccination of milk producing mammals againsttick-borne encephalitis virus infection. Still another embodiment of thepresent invention encompasses an immunogenic composition comprising thechimeric recombinant flavivirus described above in a pharmaceuticallyacceptable carrier.

The present invention also provides a method of preventing TBEVinfection in a mammal, comprising the step of administering to themammal an effective TBEV-preventing amount of a chimeric recombinantflavivirus, the chimeric flavivirus comprising a first region of nucleicacid operably encoding C, preM and E structural proteins of Langatvirus, or C protein of the mosquito-borne flavivirus plus preM and Estructural proteins of Langat virus, operably linked to a second regionof nucleic acid operably encoding non-structural proteins of amosquito-borne flavivirus, in a pharmaceutically acceptable carrier.Preferably, the mammal is a human. Advantageously, the administeringstep is intranasal, intradermal, subcutaneous, intramuscular, orintravenous. In one aspect of this preferred embodiment, the effectiveTBEV-preventing amount is between about 1 μg and 1,000 μg. The methodmay further comprise administering one or more booster injections of thechimeric flavivirus.

The present invention also contemplates a method of stimulating animmune response directed against the chimeric recombinant flavivirusesdiscussed above, in a mammal, comprising the step of administering tothe mammal an effective TBEV-preventing amount of a chimeric recombinantflavivirus, the chimeric flavivirus comprising a first region of nucleicacid operably encoding C, preM and E structural proteins of Langatvirus, or C protein of the mosquito-borne flavivirus plus preM and Estructural proteins of Langat virus, operably linked to a second regionof nucleic acid operably encoding non-structural proteins of amosquito-borne flavivirus, in a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a sequence alignment of the 5′ terminal noncoding region(NCR) sequences of the attenuated tick-borne flavivirus LGT strain TP21(SEQ ID NO: 1), its more attenuated derivative LGT E5 strain (SEQ ID NO:2) and the virulent TBEV strains Sofjin (TBEVS; SEQ ID NO: 3) andNeudoerfl (TBEVN; SEQ ID NO: 4) and the virulent North American StrainPowassan (POW; SEQ ID NO: 5). Nucleotides identical to the sequence ofLGT strain E5 are depicted by dots, and gaps introduced for thealignment are shown by dashes. Sequence numbers are shown to the right.Initiation and stop codons are underlined.

FIG. 1B shows a sequence alignment of the 3′ terminal-NCR sequences ofthe attenuated tick-borne flavivirus LGT strain TP21 (SEQ ID NO: 6), itsmore attenuated derivative LGT E5 strain (SEQ ID NO: 7) and the virulentTBEV strains Sofjin (TBEVS; SEQ ID NO: 8) and Neudoerfl (TBEVN; SEQ IDNO: 9) and the virulent North American strain Powassan (POW; SEQ ID NO:10). Nucleotides identical to the sequence of LGT strain E5 are depictedby dots, and gaps introduced for the alignment are shown by dashes.Sequence numbers are shown to the right. Initiation and stop codons areunderlined.

FIGS. 2A-2C illustrate the growth of parental and chimeric flavivirusesin simian LLCMK₂ (FIG. 2A), simian Vero (FIG. 2B) and mosquito cells(FIG. 2C). Chimeric virus inocula were grown in mosquito cells. ParentalLGT TP21 and E5 virus inocula were grown in simian Vero cells. DEN4parental virus inoculum for mosquito cells and simian cells were grownin mosquito cells and simian Vero cells, respectively. Simian LLCMK₂ orVero cells were infected with: (i) DEN4, TP21 or E5 virus at amultiplicity of infection (MOI) of 0.01, or (ii) chimeric TP21/DEN4 orE5/DEN4 virus at a MOI of 0.5. Mosquito C6/36 cells were infected with:(i) DEN4, TP21/DEN4 or E5/DEN4 virus at a MOI of 0.01 or (ii) with TP21or E5 virus at a MOI of 1,000. Cells were harvested at the indicated dayafter infection and virus titer was determined by a plaque assay on thesame cells used for study of virus replication. Plaques were counted 7or 8 days post-infection. Reduction in plaque size indicates that theviruses exhibit a growth-restriction phenotype.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes the observation that LGT/DEN4 chimericviruses containing the preM and E structural proteins of LGT strainsTP21 or E5 exhibited restriction of growth and plaque formation wheninoculated into simian cell cultures. E5 is a more attenuated derivativeof TP21 (Thind et al., Amer. J. Epidemiol. 84:198-213, 1966) whichdiffers therefrom by 24 nucleotides which produces 11 coding changes,four of which are in the E protein (Table 2). These LGT/DEN4 chimeraswere at least 5,000 times less neurovirulent than parental LGT virusesin suckling mice (Table 3). Also, these chimeras lacked detectableevidence of neuroinvasiveness following intraperitoneal inoculation of10⁵ plaque forming units (PFU) in Swiss mice or 10⁷ PFU in SCID mice. Incontrast, TP21 or E5 had intraperitoneal LD₅₀ values in SCID mice of0.004 and 0.06 PFU, respectively. Although the chimeras lackeddetectable neuroinvasiveness, even in SCID mice, intraperitonealinoculation of 10 or 10³ PFU of either virus induced LGT neutralizingantibodies and resistance to fatal encephalitis caused by LGT TP 21challenge. Thus, the LGT/DEN4 construct is useful as a vaccine for anystrain of TBEV, including the Eastern and Western subtypes, all of whichare closely related antigenically. Although DEN4 was used as amosquito-borne flavivirus to produce the chimeric vaccine, the use ofother strains of Dengue virus (i.e. types 1, 2 and 3) is alsocontemplated due to the high level of sequence homology and closeantigenic relationship of these other types with type 4. Further, theuse of a chimeric vaccine comprising any mosquito-borne flavivirusnonstructural protein cDNA (including yellow fever virus), optionallyincluding the C protein from a mosquito-borne flavivirus or from Langatvirus, in combination with LGT cDNA encoding the preM and E proteins isalso within the scope of the present invention. These chimericconstructs include point mutations, insertions, deletions andsubstitutions in any of the viral genes which does not compromise theability of the construct to provide protective immunity againstchallenge with the virulent TBEV parental virus.

The complete nucleotide sequence was determined for the genome of wildtype LGT virus (TP21 strain) and its more attenuated E5 derivativeproduced by multiple passages in chick embryo tissue. Full-length DEN4cDNA was used to engineer chimeric LGT/DEN4 expression vector constructsby substituting the structural or nonstructural protein genes of LGTTP21 or LGT E5 for the corresponding DEN4 genes using methods well knownin the art of molecular biology which are described in such reference asSambrook et al. (Molecular Cloning: a Laboratory Manual, Cold SpringHarbor Press, Cold Spring Harbor N.Y., 1988; Ausubel, Current Protocolsin Molecular Biology, 1989). Only two of the 16 chimeric constructstested (depicted in Table 1A) were viable, as judged by their ability togrow and produce viral particles in culture (Table 1B). These twochimeric viruses contained the preM and E genes of LGT virus strain TP21or strain E5 and all other sequences from DEN4. These chimeras differedin their LGT-derived sequences at only four amino acid positions in theE protein (Table 2). The chimeric TP21/DEN4 and E5/DEN4 viruses wereanalyzed for their efficiency of growth in cell culture, mouseneurovirulence and neuroinvasiveness (peripheral neuroinvasiveness),immunogenicity and protective efficacy. These properties were comparedto those of their LGT parents as well as those described previously forthe highly virulent, closely related TBEV.

Chimerization of LGT TP21 or LGT E5 with mosquito-borne DEN4significantly reduced replicative capacity of the resulting virus insimian cells compared to either parental virus. Significant reduction inneurovirulence was also observed when TP21 or E5 was chimerized withDEN4, suggesting that this might be a general phenomenon for viruses ofthe tick-borne flavivirus group. Thus, the chimeric viruses appeared toretain the very low neurovirulence of their mosquito-borne DEN4 parentrather than the higher mouse neurovirulence of their tick-borne LGTvirus parent.

The present invention also relates to a recombinant chimeric DNAconstruct comprising a DNA fragment encoding LGT virus preM and Eproteins and dengue virus nonstructural proteins, and a vector. This DNAfragment may encode the wild type proteins, or mutant proteins thereofincluding point mutations, insertions, deletions and the like which donot compromise the safety and efficacy of the vaccine produced from thechimeric construct. The safety and efficacy of any such chimeric viruscan be determined using the methods described herein without undueexperimentation.

In another embodiment, the invention relates to a chimeric vaccine forhumans against TBEV, comprising DNA encoding LGT wild type TP21 or E5preM and E proteins and dengue virus nonstructural proteins. Thesechimeric vaccines are evaluated in non-human primates for: (1)replicative efficiency as measured by extent and duration of viremia;(2) virulence as measured by neurologic signs following directintranasal or intracerebral inoculation or peripheral inoculation; (3)immunogenicity as indicated by the type and magnitude of antibodyresponse following viral infection, satisfactory immunogenicity andprotective efficacy; and (4) protective efficacy as measured byresistance to challenge with virulent tick-borne encephalitis virusfollowing immunization. Chimeric vaccines that show markedly reducedvirulence but retain sufficient immunogenicity in monkeys are evaluatedduring clinical trials in humans.

For use as a vaccine, the chimeric flaviviruses of the invention areformulated with a pharmaceutically acceptable carrier and parenterallyadministered to a mammal, preferably a human. Pharmaceuticallyacceptable means that the agent should be acceptable in the sense ofbeing compatible with the other ingredients of the formulation as wellas non-injurious to the patient. Such carriers include phosphatebuffered saline (PBS) and lactate Ringer's solution. Contemplated modesof administration of the vaccine include intradermal, intravenous,subcutaneous and any other route which allows entry of the vaccine intothe body at a peripheral site, i.e. skin, muscle, subcutaneous tissue,etc. The amount of the vaccine preparation administered to a mammal istypically between about 1 μg and 1,000 μg, preferably between about 50μg and 500 μg. The method may further comprise administering one or morebooster injections of the chimeric flavivirus in an amount between about1 μg and about 1,000 μg. Although the precise amount of recombinantvirus will vary depending on the individual, this amount can beoptimized using routine dose-response experiments well known to those ofordinary skill in the art.

LGT and DEN4 flaviviruses were obtained, chimerized and evaluated forneurovirulence and protective efficacy against parent LGT viruschallenge as described in the examples provided below.

EXAMPLE 1 Virus Sources and Isolation of Viruses

LGT wild type strain TP21 was originally isolated from ticks in Malayain 1956 (Gordon-Smith, Nature 178:581-582, 1956). It was then passaged11 times in mouse brain and twice in simian Vero cells. The LGT TP21strain used herein was obtained from Dr. R. Shope (Yale University, NewHaven, Conn.) from the Rockefeller Foundation Collection. The LGTattenuated strain E5 was derived from the TP21 strain by 42 passages in7-day-old chick embryos and had an additional passage in simian Verocells (Thind et al., Amer. J. Epidemiol. 84:198-213, 1966; Thind et al.,Amer. J. Epidemiol. 84:214-224, 1966). This virus was obtained from Dr.J. Huggins (USAMRIID, Frederick, Md.). Langat viruses were plaquepurified three times on Vero cells under soft agar prior to preparationof virus stocks that titered 2×10⁹ plaque forming units (pfu)/ml forTP21 virus and 1.2×10⁹ pfu/ml for E5 virus. Vero cells were grown at 37°C. in Eagle's minimum essential medium (MEM) plus 10% fetal calf serum(FCS) and incubated in an atmosphere of 5% CO₂. DEN4(clone 2A) virusrescued from the full-length cDNA construct of dengue type 4 strain814669 genome was used as the parental DEN4 virus (Lai et al., Proc.Natl. Acad. Sci. U.S.A. 88:5139-5143, 1991). All of these viruses aregenerally available from the scientific research community.

EXAMPLE 2 Genome Sequences of Langat Viruses

Purification of LGT and isolation of its RNA were performed as describedpreviously for TBEV (Pletnev et al., Virology 174:250-263, 1990). Firststrand cDNA was synthesized using the reverse transcriptase SuperScript™purification system (GibcoBRL, Life Technologies) and a syntheticoligonucleotide primer complementary to the conserved 22 3′-terminalnucleotides of TBEV strains (Mandl et al., J. Virol. 65:4070-4077, 1991;Wallner et al., Virology 213:169-178, 1995) or the same 3′-terminalsequence of Powassan virus genome (Mandl et al., Virology 194:173-184,1993). The polymerase chain reaction (PCR) was used to amplify threeoverlapping cDNA fragments of the LGT TP21 or LGT E5 genome. Thesequences of the PCR primers were derived from the published codingregion of LGT TP21 (Iacoco-Connors et al., Virology 188:875-880, 1992;Mandl et al., Virology 185:891-895, 1991). The PCR fragmentcorresponding to the 5′-noncoding region was generated using a primerwhich contained the first 21 conserved 5′-terminal nucleotides of theTBEV genome (Dobrikova et al., Bioorg. Chem. 21:528-534, 1995; Mandl etal., Virology 166:197-205, 1988; Mandl et al., J. Virol. 65:4070-4077,1991). All upstream primers contained a PvuI cleavage site. Each PCRproduct was cloned in E. coli BD1528 using the p5′-2(NotI, XhoI,□HindIII) vector (Cahour et al., Virology 207:68-76, 1995). The completenucleotide sequence of the TP21 and E5 genomes was determined bysequencing three overlapping cDNA clones on both DNA strands by thedideoxynucleotide chain termination method (Sanger et al, Proc. Natl.Acad. Sci. U.S.A., 74:5463-5467, 1977). Several independent clones foreach third of the genome were sequenced.

Sequence analysis revealed that the TP21 or E5 genome was 10, 940 or10,941 nucleotides in length, respectively, and contained a single openreading frame encoding a 3,414 amino acid polyprotein. The sequence datawere used to assess the relationship of LGT TP21 or E5 virus to othermembers of the flavivirus genus. The overall protein sequence homologybetween LGT and TBEV Far Eastern subtype (strain Sofjin) was about84.2%. In contrast, the overall sequence homology between LGT and DEN4,a mosquito-borne flavivirus, was only 39.4%. Among the tick-borne LGTand TBEV flaviviruses, the structural C, preM and E proteins are theleast conserved (74%, 75% and 88% homology, respectively), whereas thenonstructural proteins exhibit greater sequence conservation (90-95%).

FIGS. 1A-1B show the 5′- and 3′-noncoding region (NCR) sequences of LGTE5 and TP21 viruses compared to the previously published sequences ofPowassan virus, TBEV European subtype (prototype strain Neudoerfl) andFar Eastern subtype (strain Sofjin) viruses. The LGT E5 5′NCR is 130nucleotides in length and differs from the 5′NCR of TP21 virus bydeletion of a G nucleotide at position 61 and by the presence of a Cnucleotide instead of a G at position 35. Alignment of the 5′NCR of TBEcomplex virus genomes shows that two domains between nucleotides 1-30and 82-129 are conserved in the corresponding regions of TBEV, LGT andPowassan (POW) virus. Sequence between these domains is hypervariable,and the POW and LGT genomes sustained a deletion in this region comparedto TBEV strains. Among the tick-borne flaviviruses, the 3′ noncodingsequence varies significantly in length: LGT E5 or TP21 contains 566nucleotides, POW contains 480 nucleotides and TBEV strains contain350-750 nucleotides.

The alignment of 3′NCRs (FIG. 1B) revealed that the last 95 3′-terminalnucleotides were highly conserved among all sequenced tick-borneflaviviruses. Only four nucleotides in this region distinguish LGT E5 orTP21 virus from TBEV Far Eastern strain. Variation in length of the3′NCR of tick-borne flaviviruses was observed primarily between the stopcodon and the last 325 3′-terminal nucleotides, the latter being aregion that exhibits a high degree of sequence conservation. The LGTgenome has: (i) a 172 or 80 nucleotide insertion in this region comparedto the TBEV Far Eastern strain or POW and (ii) a 182 nucleotide deletioncompared to the TBEV European subtype strain. The 3′NCR of LGT E5 straindiffered from its LGT TP21 parent by the insertion of a dinucleotide(AC) between positions 10,515 and 10,516 and by deletion of C and U atposition 10,599 and 10,633, respectively.

The complete sequence of the LGT TP21 parent was compared to that of itsmore attenuated LGT E5 derivative in an attempt to identify potentialgenetic determinants of neuroinvasiveness and neurovirulence. Thisanalysis revealed 24 nucleotide differences, eleven of which resulted inan amino acid substitution in the corresponding polyprotein (Table 2).Amino acid changes were located in the C, E, NS1, NS2A and NS3 proteins.Sequence analysis of separate TP21 cDNA clones which encoded the Eprotein revealed that a C₁₄₃₆>U was present in three of four clones.Interestingly, mutation Asn₆₆₈>Asp in the E protein of strain E5corresponds to a substitution observed previously in the E protein of apartially attenuated mutant of TBEV (Holzmann et al., J. Virol.64:5156-5159, 1990; Mandl et al., J. Virol. 63:564-571, 1989). Toidentify the relative importance of the 11 amino acid differences in thevirulence of the LGT TP21 and E5 strains, different combinations of LGTvirus genes were introduced into the DEN4 cDNA genome by replacingcorresponding DEN4 genes as described below.

TABLE 1A SEQ ID Construct Amino acid/nucleotide sequences NO.pLGT(CME)/DEN4         M A G K   G L N S R N T 14AGAGAGCAGATCTCTGGAAAAATGGCCGGGAAG... ...GGCTTGAACTCGAGGAACACT 15         BglII            Xhol pLGT(ME)/DEN4 I L Q R R G S R R T   G L NS R N T 16 1. ATCCTGCAGCGCCGAGGAAGTAGAAGGACG... ...GGCTTGAACTCGAGGAACACT17         PstI           XhoI L N G R K R S I I D   G L N S R N T 18 2.CTGAACGGGAGAAAAAGATCGATCATTGAC... ...GGCTTGAACTCGAGGAACACT 19             ClaI       XhoI L N G R K R S A V D   G L N S R N T 20 3.CTGAACGGGAGAAAAAGGTCTGCAGTTGAC... ...GGCTTGAACTCGAGGAACACT 21             PstI       XhoI M A F S L V A R E R   G L N S R N T 22 4.ATGGCGTTTTCCTTGGTTGCAAGAGAGAGA... ...GGCTTGAACTCGAGGAACACT 23            BstBI       XhoI pLGT(NS1,2A)/DEN4 G T N S R N P T   R G R RS W P L N 24 GGCACGAACTCGAGGAACCCAACC... ...AGGGGGAGACGATCGTGGCCTCTTAAC25          XhoI         PvuI pLGT(NS1,2A,2B,d3)/DEN4 G T N S R N PT   G T G W I R K K R 26 GGCACGAACTCGAGGAACCCAACC......GGCACGGGCTGGATTCGAAAGAAAAGA 27          XhoI          BstBIpLGT(NS2B,3)/DEN4 G A S R R S F N E   S G R R S I T L 28GGAGCCTCAAGACGATCGTTTAATGAG... ...TCTGGAAGAAGATCTATAACTCTC 29           PvuI        BglII

TABLE 1B SEQ ID NO: LGT cDNA Viability 15  129-2379 No 17  403-2379 No19  426-2379 No 21  428-2379 Yes 23  490-2379 No 25 2382-4205 No 272382-5161 No 29 4201-6456 No Note: The terminal sequences of thecorresponding cDNA fragments of LGT TP21 and LGT E5 are identical.Restriction enzyme-cleaved LGT TP21 or E5 cDNA fragments were insertedinto DEN4 cDNA at appropriate sites as indicated by the underlinedsequence. The amino acid and the encoding nucleotide sequences of LGTTP21 are in bold letters. Infectivity of RNA transcripts from DNAconsructs was tested by transfecting simian or mosquito cells andevaluating cell cultures for evidence of # infection byimmunofluourescence assay (IFA).

EXAMPLE 3 Construction of LGT/DEN4 Chimeras and Viability Determination

Chimeric LGT/DEN4 viruses were constructed to analyze the genetic basisfor reduced neurovirulence, tissue tropism and peripheral invasivenessof Langat virus and to develop a safe and effective live attenuatedvirus vaccine against the antigenically related TBEV. Full-length DEN4cDNA was used to engineer chimeric constructs containing the LGTC-preM-E, preM-E, NS1-NS2A, NS1-NS2A-NS2B-dNS3 or NS2B-NS3 genes withthe remaining sequences being derived from DEN4 (Table 1A). In eachinstance, the terminal sequences of the corresponding cDNA fragments ofLGT TP21 and LGT E5 used to construct the chimeras were identical.

TABLE 2 Strain E5 TP21 Gene NT, AA NT, AA C C₃₇₁ Pro₈₀ A Thr A₄₆₁ Ile₁₁₀C Leu preM U₅₁₄ G E A₁₃₂₇ C A₁₃₄₂ G C₁₄₃₆ Thr₄₃₅ C/U Thr/Ile U₁₅₆₇ AA₁₈₂₃ Ser₅₆₄ G Gly C₁₉₆₈ Ser₆₁₂ U Phe G₂₁₃₅ Asp₆₆₈ A Asn NSI A₃₀₀₈Met₉₆₀ G Val U₃₄₀₃ C NS2A G₃₆₃₅ Ala₁₁₆₈ C Pro C₃₆₃₇ G A₃₉₆₄ G NS3 G₄₆₆₂Ser₁₅₁₀ A Asn A₅₃₃₉ Tyr₁₇₃₆ U Phe U₅₃₇₄ C C₅₅₄₆ Leu₁₈₀₅ U Phe Note: NTor AA indicates nucleotide or amino acid residue at indicated positionof LGT E5 genome or polyprotein. Polyprotein sequence of strain TP21determined in this study differed from that of the TP21 polyproteinpublished earlier (Iacoco-Connors et al., Virology 188:875-880, 1992;Mandl et al., Virology 185:891-895, 1991). Glu-152, Ser-564, Ser-612,Met-960, Ala-1168, Ile-2149, Val-2357, Ser-2775, Ala-2921, Cys-3157 andVal-3158 were replaced by Asp, Gly, Phe, Val, Pro, Met, # Ile, Cys, Gly,Trp and Leu residues, respectively.

Plasmid DEN4 p2A(XhoI) (Bray et al., Proc. Natl. Acad. Sci. U.S.A.88:10342-10346, 1991) and pTBEV(ME)/DEN4 (Pletnev et al., Proc. Natl.Acad. Sci. U.S.A. 89:10532-10536, 1992) were used to substitute two ormore LGT genes for the corresponding DEN4 genes.Oligonucleotide-directed mutagenesis was performed to introduce a ClaIsite instead of the unique Asp718 site at the 3′ end of dengue sequencein p2A(XhoI). To facilitate construction of chimeric LGT TP21(CME)/DEN4or LGT E5(CME)/DEN4 cDNA (Table 1A), the cDNA region encoding the DEN4C, preM, and E genes, extending from BglII (nucleotide 88) to the XhoIsite (nucleotide 2342) was replaced with the corresponding sequence ofTP21 or E5 virus. To construct chimeric LGT(ME)/DEN4 cDNA containing thepreM and E genes of the Langat wild type TP21 strain or its moreattenuated E5 derivative, four different junctions between the DEN4 Cgene and LGT preM gene were created in chimeric DNA plasmids (Table 1A).For example, construct number 3 of pLGT(ME)/DEN4 was prepared asfollows. The PCR fragment which contains preM-E genes between theintroduced PstI site of TP21 at nucleotide 422 or E5 at nucleotide 423and the XhoI site (TP21 nt 2379 or E5 nt 2380) near the 3′ end of the Egene was inserted into the DEN4 vector replacing the corresponding DEN4sequence, yielding a chimera similar to that containing the preM and Egenes of TBEV described previously (Pletnev et al., Proc. Natl. Acad.Sci. U.S.A. 89:10532-10536, 1992). Sequences at the junctions betweenLGT and DEN4 genes in each chimeric plasmid were verified by sequencingacross the regions. Functional integrity of the chimeric viruses wasdemonstrated by their ability to direct protein synthesis using a rabbitreticulocyte lysate or in a T7-vaccinia virus transient expressionsystem (Elroy-Stein et al., Proc. Natl. Acad. Sci. U.S.A. 86:6126-6130,1989).

EXAMPLE 4 Growth of Chimeras in Cell Culture

Full-length RNA transcripts made from the chimeric cDNA templatesdescribed above were tested for infectivity by transfecting simianLLCMK₂, simian Vero and mosquito C6/36 cells in the presence of DOTAP(Pletnev et al., supra.). Nine days after transfection, cells in a24-well plate were transferred to a 6-well plate and a chamber slide. Onday 12 and again on days 16, 20, 24, 28, 32, 36, and 44, cells weresplit and passaged. In addition, cells were examined on each of thesedays by immunofluorescence assay (IFA) for the presence of DEN4 and LGTantigens using a 1 :300 dilution of DEN4- or LGT-specific hyperimmunemouse ascitic fluid (HMAf). An infectious virus was only recovered fromtwo of the 16 constructs (8 of LGT TP21/DEN4 and 8 of LGT E5/DEN4) shownin Table 1B and only in mosquito cells. These chimeric viruses aredesignated: (i) TP21/DEN4 for LGT wild type TP21 strain/DEN4 chimerawhich contains LGT TP21 preM and E genes; and (ii) E5/DEN4 for LGTattenuated E5 strain/DEN4 chimera which contains LGT E5 preM and Egenes. When IFA indicated that 70-100% of cells were infected, cells inthe 6-well plate were mixed with a two-fold excess of uninfected cells,and the resulting mixture was inoculated into a 75-cm² flask which wasincubated for 7 days. The infected cells were harvested together withthe medium, mixed with an equal volume of fetal bovine serum (FBS),frozen at −70° C. and used later as seed to prepare suspensions ofprogeny virus. The titer of such virus suspensions was determined byplaque assay on mosquito C6/36 cells.

TP21/DEN4 and E5/DEN4 chimeras were amplified once in mosquito C6/36cells in a 75-cm² flask. Viral RNA was then isolated and reversetranscribed with oligo 2634 which is complementary to the DEN4 sequencefrom nucleotides 5090-5110 (SEQ ID NO: 11). Single-stranded cDNA wasused as a PCR template using with the primer pairs oligo 239 (SEQ ID NO:12) and oligo 444 (SEQ ID NO: 13). The PCR products were digested withHindIII and BamHI and cloned into the pGEM3 vector. Sequence of theLGT/DEN4 junction sites was confirmed by direct analysis of the clonedDNA inserts.

During the initial characterization of the TP21/DEN4 and E5/DEN4chimeras, sequence analysis confirmed the junction sequences betweenDEN4 and LGT preM genes or LGT E and DEN4 NS1 genes. Importantly, thechimeras differed in their LGT-derived sequences at only four amino acidpositions in the E protein (Table 2). In addition, immunoprecipitationof viral proteins from infected mosquito cell culture indicated thatboth of these chimeras produced the expected proteins. The LGT preMprotein of TP21/DEN4 or E5/DEN4 was sensitive to digestion withendoglycosidase F or H, whereas the E protein was resistant to digestionby these enzymes. This indicated that LGT E protein expressed by eitherchimera in mosquito cells was not glycosylated despite the presence of apotential glycosylation site in the E sequence.

The LGT TP21 and E5/DEN4 chimeras were compared to each other and totheir parental viruses with respect to pattern of replication andmaximum yield in simian and mosquito cells (FIG. 2). When inoculated ata multiplicity of infection (MOI) of 0.01, the chimeras grew to amoderate titer in mosquito cells, namely 10^(4.8) to 10^(6.0) PFU/ml. Incontrast, the growth of their parent LGT TP21 or E5 virus in mosquitocells was totally restricted when cell culture harvests were assayed byplaque assay on mosquito cells. This restriction was observed eventhough a MOI of 1,000 was used. Also, viral replication was not detectedwhen less LGT parental virus (ranging from a MOI of 0.01 to 100) wasused for inoculation of mosquito cells. In addition, IFA failed todetect evidence of viral replication in any of these instances. Thetiter attained by the chimeras in mosquito cells on day 5 was 10- to100-fold reduced compared to DEN4. Unlike DEN4, the two chimeras induceda chronic non-cytopathic infection of mosquito cells. Also, compared toDEN4, the two chimeras produced smaller plaques. Plaque size averaged5.0 mm for TP21/DEN4,2.0 mm for E5/DEN4 and 11.5 mm for DEN4.

A different hierarchy of viral replication was observed when simiancells were analyzed as cell substrates. Chimerization of LGT TP21 or E5with DEN4 significantly reduced the efficiency of viral replication insimian cells compared to parental LGT TP21 or E5 virus as well as DEN4.Consequently, the LGT/DEN4 chimeric viruses grown in mosquito cells wereunable to produce plaques in simian cells. In addition, these chimerasdid not replicate efficiently in simian cells as indicated by plaqueassay of growth yield in mosquito cells. In contrast, parental LGT TP21or E5 was able to produce plaques with high efficiency and grow to hightiter in simian cells, i.e. approximately 10⁸ to 10⁹ PFU/ml, a levelgreater than that achieved by DEN4.

Simian cells were not completely refractory to the chimeric virusesbecause virus propagated in permissive mosquito cell culture didinitiate slow and partially restricted viral replication in simianLLCMK₂ or Vero cells inoculated at a MOI of 0.5. In addition, spread ofvirus in these cell cultures differed from that of parental LGT TP21 orE5 virus that were cytopathic and attained a high titer by day 5 wheninoculated at a MOI of 0.01. In contrast, infection of simian cells witheither chimera at a MOI of 0.5 did not produce cytopathic effects andprogressed very slowly as monitored by IFA. An incubation period of 24to 48 days was required for 80-100% of simian cells to become infected.At this time, a chronic infection without cytopathic effect becameestablished and such chronically infected cells could be maintainedduring incubation and subculture for 10 months without apparent visibleeffect. Virus yield from these simian cell cultures at the time ofmaximum infection was measured by plaque assay on mosquito cells andfound to be reduced by 90% from the level attained when either chimericvirus was grown in mosquito cells. The yield of either chimeric virusfrom infected simian LLCMK₂ cells did not produce plaques on thesecells. This was also the case for the LGT E5/DEN4 chimera grown insimian Vero cells. In contrast, a reduced number of very small faintplaques was produced by the LGT TP21/DEN4 chimera in these cells, afinding consistent with the limited replication of the chimera in simiancells. When chimeric TP21/DEN4 or E5/DEN4 virus harvested from simiancells chronically infected for 8 months was passaged in mosquito cellculture and attained a high titer, the virus yield retained itsrestriction of growth and plaque formation in simian cells.

These results indicate that chimerization of LGT TP21 or LGT E5 withDEN4 significantly reduced the replicative capacity of there chimeras insimian cells compared to either parental virus. This is in contrast toprevious results showing that the chimera TBEV(ME)/DEN4 replicated 1,000times more efficiently in simian cells than did DEN4 (Pletnev et al.,supra.).

The neurovirulence and neuroinvasiveness of TP21, E5 or LGT/DEN 4chimeric virus was studied in an in vivo mouse model as described inExamples 5 and 6 below.

EXAMPLE 5 Neurovirulence Studies

The neurovirulence of LGT TP21, LGT E5, DEN4 and chimeric TP21/DEN4 orE5/DEN4 viruses was evaluated in 3-day-old outbred Swiss mice. Groups of10 to 14 mice were inoculated intracerebrally (IC) with decimaldilutions of virus ranging from 10⁻² to 10⁷ PFU in 20 μl modifiedEagle's medium (MEM) containing 0.25% human serum albumin. Mice wereobserved for 28 days for non-fatal or fatal encephalitis.

Wild type LGT TP21 was highly neurovirulent as shown by its LD₅₀ of 0.4PFU in suckling mice, i.e. one PFU was lethal for suckling mice (Table3). Neurovirulence of the more attenuated LGT strain E5 by the sameroute was 50 times less. DEN4 was even less neurovirulent, as disease ordeath was observed only when suckling mice were inoculated with a highdose. Intracerebral LD₅₀ of DEN4 was estimated to be 8,000 PFU. ChimericTP21/DEN4 or E5/DEN4 exhibited a significant reduction in neurovirulencecompared to its LGT parent when tested by direct inoculation into thebrain of suckling mice. Thus, the intracerebral LD₅₀ of TP21/DEN4 was2,500 PFU which was about 6,250 fold less than its LGT TP21 parent (0.4PFU). E5/DEN4 was even more attenuated, having an IC LD₅₀>10⁵ PFU. Inaddition, E5/DEN4 was 44 times less neurovirulent than TP21/DEN4. Thus,the TP21/DEN4 or E5/DEN4 chimeric virus retained the low mouseneurovirulence of its DEN4 parent rather than the higher mouseneurovirulence of its Langat virus parent. Likewise, the LGT/DEN4chimeras were at least 6,250 times less neurovirulent in mice than theirparental LGT TP21 or E5. This significant reduction of neurovirulenceappeared to correlate with the restricted growth of these chimeras insimian cells.

Surprisingly, there was a dramatic reduction in neurovirulence of theLGT TP21(ME)/DEN4 and LGT E5(ME)/DEN4 chimeras compared to theTBEV(ME)/DEN4 (Table 3). The TP21/DEN4 and E5/DEN4 chimeras were 125 and5500 times less neurovirulent, respectively, than the TBEV chimera.

TABLE 3 LD₅₀ (PFU) in Mice Immunocompetent Fold- Fold- reductionreduction vs. vs. SCID Virus Intracerebral TBEV Intraperitoneal TBEVIntraperitoneal DEN4 8 × 10³ 8 × 10⁴ >10⁷ >7.1 × 10⁵ >10⁷ TBEV 10⁻¹ —1.4 × 10¹ — NT TBEV 2 × 10¹ 200 >10⁷ >7.1 × 10⁵ NT (ME)/DEN4 LGT TP21 4× 10⁻¹  4 5 × 10³ 357 4 × 10⁻³ LGT 2.5 × 10³ 2.5 × 10⁴ >10⁵ >7.1 × 10³>10⁷ TP21/DEN4 LGT E5 2 × 10¹ 200 >10⁷ >7.1 × 10⁵ 6 × 10⁻² LGT E5/DEN41.1 × 10⁵ 1.1 × 10⁶ >10⁵ >7.1 × 10³ >10⁷ 50% lethal dose of virus (LD₅₀)was estimated by intracerebral inoculation of 3-day-old Swiss mice or byintraperitoneal inoculation of 3-week-old Swiss or SCID mice withdecimal dilutions of virus. LD₅₀ of TP21 or E5 virus is expressed as PFUmeasured in Vero cells and LD₅₀ of DEN4, TP21/DEN4 or E5/DEN4 virus isexpressed as PFU measured in C6/36 cells. LD₅₀ of TBEV and TBEV(ME/DEN4was determined previously and presented here for purpose of comparison #(Pletnev et al., Proc, Natl. Acad. Sci. U.S.A. 89:10532-10536, 1992;Pletnev et al., J. Virol. 67:4956-4963, 1993). NT, Not tested.

EXAMPLE 6 Neuroinvasiveness Studies

Neuroinvasiveness (peripheral neurovirulence) of parental or chimericviruses was evaluated in 3-week-old female Swiss mice that wereinoculated intraperitoneally (IP) in groups of ten with: (i) 10, 10²,10³, 10⁴, 10⁵, 10⁶ or 10⁷ PFU of LGT TP21 or LGT E5 virus; or (ii) 10⁵PFU of TP21/DEN4, E5/DEN4 or DEN4 virus. Mice were observed for 21 daysand surviving mice were bled to evaluate antibody response. Survivingmice were challenged IP on the next day with 100 or 1,000 IP LD₅₀ ofparental TP21 virus and observed for an additional four weeks.

LGT TP21 was also moderately virulent for 3-week-old mice wheninoculated by a peripheral route. The IP LD₅₀ was approximately 5×10³PFU. In contrast, chimeric TP21/DEN4 exhibited lower peripheralneuroinvasiveness for adult mice with an IP LD₅₀ of >10⁵ PFU. Theattenuated LGT E5 strain exhibited much lower peripheral virulence thanits LGT TP21 parent. Only 10-20% of adult mice inoculated IP with 10⁷PFU of LGT E5 showed symptoms of encephalitis which made it difficult todetect an effect of chimerization on neuroinvasiveness. However, thedata regarding LGT TP21/DEN4 indicates that chimerization of LGT TP21with DEN4 reduced or eliminated neuroinvasiveness of LGT for mice.

A more sensitive assay for neuroinvasiveness involved inoculation ofimmunodeficient (SCID) mice. Although SCID mice lack mature B and Tlymphocytes, they do have normal innate immune functions, includingfunctional macrophages, normal to elevated NK cell functions andelevated hemolytic complement activity. In this assay, female 3-week-oldCB-17 ICR/scid/scid mice (Bosma et al., Nature 301:527-530, 1983) ingroups of 5 were inoculated IP with: (i) 10⁵ or 10⁷ PFU of DEN4,TP21/DEN4 or E5/DEN4 virus or (ii) decimal dilutions of LGT TP21 or LGTE5 ranging from 10⁻⁴ to 10⁷ PFU. These mice were then observed formortality for 6 weeks.

The ability of immunodeficient SCID mice to detect neuroinvasiveness ofTP21 or E5 was approximately 10^(6.3) to 10^(8.8) greater than wasobserved for immunocompetent Swiss mice (Table 3). A markedly increasedsusceptibility to fatal disease was noted when SCID mice were inoculatedIP with either of the parental LGT viruses. The LD₅₀ of LGT TP21 forimmunodeficient mice by the IP route was 0.004 PFU (Table 3). Inaddition, LGT E5 was also highly virulent for SCID mice by the IP routewith a LD₅₀ of 0.06 PFU. The incubation period until encephalitis was 12days and death occurred within the next 5 days. Like parental DEN4virus, both LGT chimeric viruses lacked detectable neuroinvasiveness forSCID mice. Mice inoculated IP with 10⁷ PFU remained healthy during the 6week post-inoculation observation interval. These findings indicate thatthe chimeric TP21/DEN4 or E5/DEN4 had completely lost theneuroinvasiveness of its LGT parent for SCID mice.

The parental TP21 virus itself or its attenuated derivative strain E5may not be suitable for us in humans as a live-attenuated vaccineagainst the TBE complex of viruses. The occurrence of vaccine-associatedencephalitis, albeit at a very low frequency (10^(−4.3)), which wasobserved during a large clinical trial of the most attenuated vaccinecandidate (Yelantsev virus) against TBEV, supports this view(Iacoco-Connors et al., Virology 188:875-880, 1992; Smorodincev et al.,in Tick-borne Encephalitis and its Vaccine Prophylaxis, Leningrad,1986). Live attenuated tick-borne flaviviruses should be free of such avery low level of virulence if possible. In contrast to parental LGTviruses, evaluation of TP21/DEN4 and E5/DEN4 chimeric viruses in theSCID mouse model indicated that the two chimeras were completelyavirulent and free of any evidence of neuroinvasiveness in this verysensitive assay system, as a large dose (10⁷ PFU) of either chimericvirus failed to invade the CNS and cause encephalitis or death followinginoculation at a peripheral site. This suggests that proteins encoded byregions of the LGT genome other than the preM and E genes are requiredfor either LGT virus to spread from a peripheral site to the brain andcause encephalitis.

Neuroinvasiveness could be differentiated from neurovirulence. Thus, LGTE5 exhibited moderate neurovirulence when virus was inoculated directlyinto the brain of suckling mice (LD₅₀ of 20 PFU) whereas its LD₅₀ wheninoculated IP was >10⁷ PFU. Clearly, neurovirulence is required for LGTto produce encephalitis when inoculated by a peripheral route such as IPinoculation. However, other properties of these flaviviruses arerequired for virus to disseminate to the brain.

EXAMPLE 7 Immunogenicity and Protective Efficacy of LGT/DEN4 ChimericViruses Against Langat Virus Challenge

Mice were used to determine the immunogenicity and protective efficacyof the LGT/DEN4 chimeras. Mice inoculated IP with 10² PFU of TP21 or E5,or of 10⁵ PFU of TP21/DEN4 or E5/DEN4, developed a high antibodyresponse to TP21 virions as measured by enzyme linked immunosorbentassay (ELISA; Table 4). In addition, these mice developed a high levelof neutralizing antibodies against LGT TP21 as measured by plaquereduction. In contrast, mice inoculated IP with 10⁵ PFU of DEN4 failedto develop TP21 or E5 neutralizing antibodies measurable by ELISA.

TABLE 4 Mean of antibody titer Dose for immu- (reciprocal) measured byVirus Nization (PFU) ELISA¹ NT-test² DEN4 10⁵  <50 <20 TP21 10² 7560 703E5 10² 7080 761 TP21/DEN4 10⁵ 2400 288 E5/DEN4 10⁵ 2320 327 ¹Antibodytiter in mouse serum collected three weeks after intraperitonealimmunization was determined using purified TP21 virions as antigen.²Neutralizing antibodies were measured by a 50% plaque reductionneutralization test using TP21 virus.

Twenty-three days after inoculation with TP21/DEN4, E5/DEN4, TP21 or E5,mice were challenged IP with 100 or 1,000 IP LD₅₀ of TP21 (Table 5). Allof the mice that had been immunized previously with TP21/DEN4 or E5/DEN4developed a high titer of neutralizing antibodies and were completelyprotected against IP challenge of TP21. In contrast, mice immunized IPwith DEN4 were only partially protected: three of ten mice died whenchallenged with 100 IP LD₅₀ of LGT TP 21. Each of the 30 nonimmunizedcontrol mice developed encephalitis and 25 subsequently died. Asobserved earlier, these results indicate that an immune response to DEN4nonstructural proteins was not able to provide complete protection formice against LGT TP21.

TABLE 5 Mortality Mortality of survivors following Infecting virusfollowing inoculation IP with TP21 Dose intraperitoneal (IntraperitonealLD₅₀) Strain (PFU) inoculation 100 1000 TP21 10² 1/10 0/9 E5 10² 0/100/10 10⁷ 1/10  0/9 DEN4 10⁵ 0/10 3/10 TP21/DEN4 10⁵ 0/40 1#/10  0/30E5/DEN4 10⁵ 0/40 0/10  0/30 Non- NA 0/30 8/10* 17/20* infected controls*Mice that survived intraperitoneal challenge with TP21 were paralyzedfor 3-5 days. # Traumatic death.

In a subsequent study (Table 6), each of 5 mice inoculated with 10⁵ PFUof DEN4 failed to resist challenge with 1,000 IP LD₅₀ of LGT TP21,whereas each of five mice immunized IP with 10⁵ PFU of chimericTP21/DEN4 or E5/DEN4 survived the same challenge with LGT TP21. Thus,the LGT E and preM proteins appear to represent the major protectiveantigens of the chimeras responsible for complete resistance to lethalLGT challenge.

TABLE 6 Dose of ELISA Mean Mortality following immu- Mean neut.challenge IP with 1000 Immunizing Nization antibody antibodyintraperitoneal LD₅₀ virus (PFU) titer titer of TP21 E5/DEN4 10  240  520/5 10³  200  52 1/5 10⁵ 1530 151 0/5 E5/DEN4- 10* <100 <20 4/5 UV 10³*<100 <20 4/5 10⁵*  100 <20 4/5 TP21/DEN4 10  240  65 1/5 10³  280 1740/5 10⁵ 3680 257 0/5 TP21/DEN4- 10* <100 <20 5/5 UV 10³* <100 <20 5/510⁵* <100 <20 3/5 TP21 10 6400 528 0/2 E5 10 6400 536 0/3 DEN4 10⁵ <100<20 5/5 Control NA <100 <20 5/5 *Titer prior to UV irradiation thatcompletely inactivated infectivity.

It is possible that the immunogenicity and protective efficacy of theparental LGT viruses and their DEN4 chimeras resulted from immunizationwith pre-formed antigens present in viral preparations inoculated IP andnot from antigens produced by virus that replicated in vivo. This issuewas addressed by evaluating antibody responses and protective efficacyof virus preparations that contained only 10 PFU. In addition, thechimeras were evaluated at two other levels of infectivity, namely 10³or 10⁵ PFU (Table 6). At each of the three doses evaluated, virus wastested without modification or after complete inactivation by UVirradiation. Prior to performing this study, the time required forcomplete inactivation of infectivity by UV was determined by kineticanalysis to be 60 minutes.

Mice were observed for 21 days, and survivors were bled 22 days afterinoculation to evaluate antibody response measured by ELISA or aplaque-reduction neutralization test. Surviving mice were challenged IPat 24 days with 1,000 IP LD₅₀ (5×10⁷ PFU) of LGT TP21 and observed forthe next four weeks.

As little as 10 PFU of either chimera regularly induced neutralizing andELISA antibodies at a titer of 1:50 or higher (Table 6), whereas only 1of 10 mice that received UV-inactivated 10⁵ PFU of a chimera developed alow level of ELISA antibodies and none developed measurable neutralizingantibodies. The response of these mice challenged with TP21 (1,000 IPLD₅₀) was consistent with the immunological response. Only 1 of 10 miceinoculated IP with 10 PFU of a chimera succumbed to challenge, whereas 7of 10 mice that received UV-inactivated 10⁵ PFU died after challenge. itappears that successful immunization by the two chimeras primarilyreflects the effect of viral replication and not the effect of a largemass of pre-formed viral antigens.

EXAMPLE 8 Immunogenicity and Protective Efficacy of LGT/DEN4 ChimericViruses Against TBEV Challenge

Mice are used to determine the immunogenicity and protective efficacy ofthe LGT/DEN4 chimeras. Twenty-three days after inoculation withTP21/DEN4, E5/DEN4, or DEN4, mice are challenged with 10³ or 10⁵ PFU ofTBEV or one of its highly virulent tick-borne flavivirus relatives, suchas Omsk hemorrhagic fever virus, Kyasamur forest disease virus, Negishivirus, Powassan virus, or Central European tick-borne encephalitisvirus. All of the mice immunized previously with TP21/DEN4 or E5/DEN4develop a high titer of neutralizing antibodies and are completelyprotected against IP challenge of TBEV. In contrast, mice immunized IPwith DEN4 are only partially protected or not protected at all. Each ofthe nonimmunized control mice develope encephalitis and die. Theseresults indicate that an immune response to DEN4 nonstructural proteinsis not able to provide complete protection for mice against TBEV or itshighly virulent relatives.

In an additional study, each of a group of mice are inoculated with 10⁵PFU of DEN4 fail to resist challenge with TBEV, whereas each of a groupof mice immunized IP with 10⁵ PFU of chimeric TP21/DEN4 or E5/DEN4survive the same challenge with TBEV. Thus, the LGT preM and E proteinsappear to represent the major protective antigens of the chimerasresponsible for complete resistance to lethal TBEV challenge.

It is possible that the immunogenicity and protective efficacy of theparental LGT viruses and their DEN4 chimeras results from immunizationwith pre-formed antigens present in viral preparations inoculated IP andnot from antigens produced by virus that replicates in vivo. This issueis addressed by evaluating antibody responses and protective efficacy ofvirus preparations that contain only limited amounts of virus. Inaddition, the chimeras are evaluated at two other levels of infectivity,namely 10³ or 10⁵ PFU. At each of the three doses, the viruses aretested without modification or after complete inactivation by UVirradiation. Prior to performing this study, the time required forcomplete inactivation of infectivity by UV is determined by kineticanalysis to be 60 minutes.

Mice are observed for 21 days, and survivors are bled 22 days afterinoculation to evaluate antibody response measured by ELISA or aplaque-reduction neutralization test. Surviving mice are challenged IPat 24 days with 1,000 IP LD₅₀ of TBEV and observed for the next fourweeks.

As little as 10 PFU of either chimera regularly induces neutralizing andELISA antibodies at a titer of 1:50 or higher (Table 6), whereas only asmall percentage of mice that receive UV-inactivated 10⁵ PFU of achimera develop a low level of ELISA antibodies and none developmeasurable neutralizing antibodies. The response of these micechallenged with TP21 (1,000 IP LD₅₀) is consistent with theimmunological response. Only a small percentage of mice inoculated IPwith 10 PFU of a chimera succumb to challenge, whereas a majority of themice that receive UV-inactivated 10⁵ PFU die after challenge. It appearsthat successful immunization by the two chimeras primarily reflects theeffect of viral replication and not the effect of a large mass ofpre-formed viral antigens.

EXAMPLE 9 Testing of Chimeric Flavivirus Vaccines in Primates

Intranasal infection of monkeys represents an infected under naturalconditions experimental system in which to test and predict the efficacyof a TBE vaccine for use in humans. Hambleton, P., et al., Infect.Immun., 40:995-1003 (1983). This report showed that the response ofrhesus monkeys to intranasal TBE infection is similar to that of humansexcept that no pyrexia was not observed. The objectives of the presentstudy concerning vaccination against TBE infection in monkeys are: (1)to evaluate the immunogenicity of various candidate chimeric recombinantflavivirus virus vaccines; and (2) to evaluate the protective efficacyof the above-mentioned vaccines against challenge by virulent strains ofTBEV or its closely related viruses.

A group of adult rhesus monkeys (Macaca mulatta) of both sexes, weighing1.8 to 5.1 kg, is used in the study. The animals are housed and fedaccording to standard protocols well known and accepted in the art. Forinfection and all sampling procedures, the animals are anesthetized byintramuscular injection with a suitable agent well known in the art,such as ketamine hydrochloride (Vetalar; Parke, Davis & Co.).

The virus strains that are used to infect the test animals are of aclinically important member of the tick-borne encephalitis complex. Forexample, the Russian spring-summer encephalitis (Far Eastern), CentralEuropean encephalitis (Western), Omsk hemorrhagic fever, louping ill,Kyasanur forest disease, Negishi, or Powassan viruses may be used totest the efficacy of the vaccines of the present invention.

The group of monkeys chosen for the study are divided randomly into anexperimental group and a control group. The experimental group ofmonkeys are vaccinated with an effective dose of a chimeric flavivirusvaccine of the present invention, while the control group of monkeysremain untreated. All monkeys are infected with 3×10⁸ to 5×10⁸ PFU of astandardized challenge stock of the chosen TBEV. The subject animals areinfected intranasally so as to produce clinical results similar to thoseobserved in humans infected with TBEV under natural conditions.

Blood (10 ml) is removed from the subject monkeys for testing atintervals after infection using standard techniques well known in theart. These blood samples are fractionated into their component portions,and the filtered serum samples are stored at −20° C. according toacceptable procedures known in the art. Cerebrospinal fluid (CSF) istaken from monkeys by cisternal puncture, or by other acceptablemethods, before infection and 9 to 11 days postinfection. These samplesare filtered and stored at −20° C. according to acceptable proceduresknown in the art. Portions of the unfiltered CSF are examinedmicorscopically for the presence of leukocytes.

Monkeys are killed at intervals from 4 days to 14 weeks after infectionand in extremis by i.v. injection of sodium pentobarbital. Necropsy iscarried out immediately. The brain and spinal cord are removed, andportions of tissue from each region are taken for the purpose of virusisolation. The remainder of the central nervous system (CNS) is fixed inbuffered 10% neutral Formalin, as are portions of liver, spleen, lung,kidney, and small intestine. After processing by standard methods andembedding in paraffin wax, sections of all the tissues are cut andstained by hematoxylin and eosin. Selected sections of the CNS are alsobe stained by phosphotungstic acid hematoxylin, by luxol fastblue-cresyl violet, and by the Glees and Marsland modification of theBielschowsky technique for neurones.

In a majority of the control monkeys infected intranasally withchallenge TBEV, there is an onset of clinical neurological signs between10 and 15 days. These signs consist of tremors of the arms, necktwisting, uncoordination, posterior paresis, and, occasionally,convulsions. These symptoms progress to coma and death 12 to 24 hoursafter the first onset.

A clinical chemistry examination of the blood and CSF indicates thechanges in the subject animals are a result of challenge with TBEV andalso provide a means to compare animal and human data. Concerning theblood: changes in the activities of some blood components such ascertain serum enzymes are apparent from about 10 days after infection.Asparatate aminotransferase (ASAT) activity may increase. Dehydrogenaseand creatine kinase activities may also increase. In contrast, alkalinephosphatase activity may decrease progressively in relation topreinfection levels. Concerning the CSF: the activity of ASAT may besignificantly elevated postinfection, however, increases in leukocytenumbers may fail to increase as a result of infection.

Other effects of viral challenge are also observed. Lesions are found inthose challenged animals that show clinical signs of disease. Theselesions are present principally in the cerebellum, posterior brain stem,and vervical spinal cord, but milder lesions may be present in thecerebral cortex and midbrain. Virus may also appear transiently in theblood of challenged animals. An antibody response by animals challengedwith TBEV intranasally may not necessarily be observed.

In contrast to the control group, the experimental group of monkeysremain substantially free of disease symptoms caused by the challengingTBEV. The protection conferred by the chimeric flavivirus vaccine to theexperimental group of monkeys demonstrates the safety and efficacy ofthe chimeric flaviviruses of the present invention.

EXAMPLE 10 A Randomized Trial of LGT/DEN4 Chimeric Virus Vaccine

A LGT/DEN4 chimeric virus vaccine produced as described in the Examplesabove is used to test the safety and efficacy of such vaccines for humanuse. This Example is based upon: the study reported by Harabacz, I., etal. Vaccine, 10(3):145-150 (1992). One dose of the virus in the vaccinecontains 1.0, 1.5 and 2.0 μg of recombinant virus particles. The trialis designed as a prospective, multicenter, controlled, double-blindstudy. Randomization of the participants is performed with regard todose and vaccination schedule. The three dosages are randomly assignedin a ratio of 1:1:1. Random allocation to the conventional orabbreviated schedule is in a ratio of 2:1. The study centers may belocated in various European countries where TBE is known to occur, suchas Germany, Yugoslavia, Czechoslovakia and Switzerland.

A group of health adults regardless of sex are enrolled in the study.The age of the participants may range from 18 to 70 years of age. Ageand sex are distributed in a balanced fashion between the groups. Allvolunteers are required to give their informed consent before enteringthe trial; the study is approved by the appropriate ethical oversightcommittees at the centers where the studies are performed.

Concerning the schedules of vaccination and observation: the‘conventional’ immunization schedule consists of vaccinations on days 0,28, and 300. Blood sampling for antibody assays occurs on days 0, 28,42, 56, 300, 314, and 328. The ‘abbreviated’ immunization scheduleconsists of vaccination on days 0, 7, and 21. Blood samples arecollected on days 0, 21, 28, 35, 49, and 321. The vaccine tested isinjected intramuscularly into the deltoid muscle. Each subject isfollowed up for 28 days after each immunization to monitor for adversevaccine events.

Antibody assays using standard immunological techniques such as theenzyme-linked immunosorbent assay (ELISA), thehemagglutination-inhibition test (HIT) and neutralization test (NT) areperformed. These assays are performed according to methods well known inthe art, for example, the HIT may be performed according to the methodof Clarke and Casals, Am. J. Trop. Med. Hyg., 7:561-573 (1958), theELISA according to Heinz, et al., Virology, 126:525-538 (1983), and theNT as described by Klockmann, et al., J. Biol. Stand., 17:331-342(1989). In NT, the ND₅₀ per 0.1 ml may be determined using a virus doseof 100 TCID₅₀ per 0.1 ml. Lower limits for seroconversion may be definedas 8 in HIT, 2 I NT, and 160 in ELISA.

The results of the study show that the chimeric flavivirus vaccines ofthe present invention are safe for use in humans. They also show thatimmunized individuals seroconvert and produce antibodies against thechimeric flavivirus vaccines. These antibodies have HIT and NT resultsthat indicate that the immune response elicited by the vaccines isprotective against TBEV challenge. This study also shows that adverseevents related to vaccination are few compared to the well toleratedreactions of a majority of immunized subjects in the study.

The above description of the invention is set forth solely to assist inunderstanding the invention. It is to be understood that variations ofthe invention, including all equivalents now known or later developed,are to be considered as falling within the scope of the invention, whichis limited only by the following claims.

29 1 174 DNA Flavivirus, Langat 1 agauuuucuu gcgcgugcau gcgugugcuucagacagccc aggcagcgac ugugauugug 60 gauauucuuu cugcaaguuu ugucgugaacguguugagaa aaagacagcu uaggagaaca 120 agagcuggga auggccggga aggccguucuaaaaggaaag gggggggguc cccc 174 2 173 DNA Flavivirus, Langat 2 agauuuucuugcgcgugcau gcgugugcuu cagagagccc aggcagcgac ugugauugug 60 auauucuuucugcaaguuuu gucgugaacg uguugagaaa aagacagcuu aggagaacaa 120 gagcugggaauggccgggaa ggccguucua aaaggaaagg gggggggucc ccc 173 3 177 DNAFlavivirus, Tick-borne Encephalitis 3 agauuuucuu gcacgugcau gcguuugcuccggauagcaa cagcagcgac agguuugaga 60 gagagacaau cuuucgcuug aucagucgugaacguguuga gaaaaagaca gcuuaggaga 120 acaagagcug gggauggccg ggaaggccauucugaaagga aaggggggcg guccccc 177 4 176 DNA Flavivirus, Tick-borneEncephalitis 4 agauuuucuu gcacgugcau gcguuugcuu cggacagcau uagcagcgguugguuugaaa 60 gagauauucu uuuguuucua ccagucguga acguguugag aaaaagacagcuuaggagaa 120 caagagcugg ggauggucaa gaaggccauc cuaaaaggua aggggggcgguccccc 176 5 149 DNA Flavivirus, Tick-borne Encephalitis 5 agauuuucuugcacgugugu gcgggugcuu uagucagugu ccgcagcguu cuguugaacg 60 ugaguguguugagaaaaaga cagcuuagga gaacaagagc ugggaguggu uaugaugacc 120 acuucuaaaggaaagggggg cgguccccc 149 6 587 DNA Flavivirus, Langat 6 cuggagagcucaauauuuua aagccagaca caaggagucc aaccuggagg gcucuugaaa 60 aacucguccagaaaccaaac aaaugagcaa gucaacagga gaugauaacu cguacgagcu 120 gaucuccaacacacaagaaa aaugguggga ugcggcaacg cgaggcucgu gacggggaaa 180 ugaucgcucccgacgcaccc cuccauugga gacaacuucg ugagaucccc cagguguuua 240 ggggcacacgccugagguaa gcaagcccca gggcgcauuc cggcagcaca ccagugagag 300 uggugacgggaaacugguca cucccgacgg acgugcgccu ugugaaacuu ugugagaccc 360 cuugcguccagagaaggccg aacugggcgu uauaaggagg ccccaggggg gaaaccccug 420 ggaggagggaagagagaaau uggcaacucu cuucaggaua uuuccuccuc cuauaccaaa 480 uguccccucgucagaggggg ggcgguucuu guucucccug agccaccauc accuagacac 540 agauagucugaaaaggaggu gaugcguguc ucggaaaaac acccgcu 587 7 587 DNA Flavivirus,Langat 7 cuggagagcu caauauuuua aagccagaca caaggagucc aaccuggagggcucuugaaa 60 aacucgucca gaaaccaaac aaaugagcaa gucaacagga gaugauaacucguacgagcu 120 gaucuccaac acacaagaaa aaugguggga ugcggcaacg cacgaggcucgugacgggga 180 aaugaucgcu cccgacgcac cccuccauug gagacaacuu cgugagaucccccagguguu 240 uaggggacac gccugaggua agcaagcccc agggcgcauc cggcagcacaccagugagag 300 uggugacggg aaacugguca cucccgacgg acgugcgccu ugugaaacuuugugagaccc 360 cuugcgucca gagaaggccg aacugggcgu uauaaggagg ccccaggggggaaaccccug 420 ggaggaggga agagagaaau uggcaacucu cuucaggaua uuuccuccuccuauaccaaa 480 uguccccucg ucagaggggg ggcgguucuu guucucccug agccaccaucaccuagacac 540 agauagucug aaaaggaggu gaugcguguc ucggaaaaac acccgcu 587 8414 DNA Flavivirus, Tick-borne Encephalitis 8 cuggagagcu caauaaucuaaaaccagacu gugacugagc aaaaccugga gugcucguua 60 aacauugucc agaaccaaaaacaaaacaca cccccggagu gcccuacggc aacacgucaa 120 ugagaguggc gacgggaacauggucgaccc cgacguaggg cauucuguua aacuuuguga 180 gacccccggc accaugauaaggccgaacau ggugcaagaa cgggaggccc ccggaagcau 240 gcuuccggga ggagggaagagagaaauugg caacucucuu cgggauuuuu ccuccuccua 300 uaccaaauuc cccuucaauagagggggggc gguucuuguu cucccugagc caccaucacc 360 cagacacaga uagucugacaaggaggugau gugugacucg gaaaaacacc cgcu 414 9 785 DNA Flavivirus,Tick-borne Encephalitis 9 cuggagagcu caauaaucua aacccagacu gugacagagcaaaacccgga aggcucguaa 60 aagauugucc ggaaccaaaa gaaaagcaag caacucacagagauagagcu cggacuggag 120 agcucuuuaa acaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa 180 agccagaauu gagcugaacc uggagagcuc auuaaauacaguccagacga aacaaaacau 240 gacaaagcaa agaggcugag cuaaaaguuc ccacuacgggacugcuucau agcgguuugu 300 ggggggaggc uaggaggcga agccacagau cauggaaugaugcggcagcg cgcgagagcg 360 acggggaagu ggucgcaccc gacgcaccau ccaugaagcaauacuucgug agaccccccc 420 ugaccagcaa agggggcaga ccggucaggg gugaggaaugcccccagagu gcauuacggc 480 agcacgccag ugagaguggc gacgggaaaa uggucgaucccgacguaggg cacucugaaa 540 aauuuuguga gacccccugc aucaugauaa ggccgaacauggugcaugaa aggggaggcc 600 cccggaagca cgcuuccggg aggagggaag agagaaauuggcagcucucu ucaggauuuu 660 uccuccuccu auacaaaauu cccccucggu agagggggggcgguucuugu ucucccugag 720 ccaccaucac ccagacacag guagucugac aaggaggugaugugugacuc ggaaaaacac 780 ccgcu 785 10 501 DNA Flavivirus, Tick-borneEncephalitis 10 cuagagagcu cgauaaucua aacuagcaug acugaacagu caaaagaacccuaacacagg 60 ggauggugug gcagcgcaca acgacaucgu gacgggagug ggucgcccccgacgcaccau 120 ccucuuggga aaaauuuucg ugagacccuc acggcuggca aagggcaccagucguguagu 180 aagaaggccc uggcccagug cggcagcaca cucagugacg ggaaaguggucgcucccgac 240 guaacugggu aaaaacgaac uuugugagac caaaaggccu ccuggaaggcucaccaggag 300 uuaggccguu uaggagcccc cgagcauaac ucgggaggag ggaggaagaaaauuggcaau 360 cuuccucggg auuuuuccgc cuccuauacu aaauuucccc caggaaacugggggggcggu 420 ucuuguucuc ccugagccac caccauccag gcacagauag ccugacaaggagauggugug 480 ugacucggaa aaacacccgc u 501 11 27 DNA Artificial SequenceSynthetic Oligonucleotide complementary to DEN4 11 gaccgacaag gacagttccaaatcgga 27 12 21 DNA Artificial Sequence Synthetic PCR primer 12gctccggggt gtaagtccat t 21 13 40 DNA Artificial Sequence Synthetic PCRprimer 13 tctctatctg ccaagtctgg atccttgagc tctctatcca 40 14 11 PRTArtificial Sequence Synthetic chimeric flavivirus protein sequence 14Met Ala Gly Lys Gly Leu Asn Ser Arg Asn Thr 1 5 10 15 54 DNA ArtificialSequence Synthetic chimeric flavivirus nucleic acid sequence 15agagagcaga tctctggaaa aatggccggg aagggcttga actcgaggaa cact 54 16 17 PRTSynthetic chimeric flavivirus protein sequence 16 Ile Leu Gln Arg ArgGly Ser Arg Arg Thr Gly Leu Asn Ser Arg Asn 1 5 10 15 Thr 17 51 DNAArtificial Sequence Synthetic chimeric flavivirus nucleic acid sequence17 atcctgcagc gccgaggaag tagaaggacg ggcttgaact cgaggaacac t 51 18 17 PRTArtificial Sequence Synthetic chimeric flavivirus protein sequence 18Leu Asn Gly Arg Lys Arg Ser Ile Ile Asp Gly Leu Asn Ser Arg Asn 1 5 1015 Thr 19 51 DNA Artificial Sequence Synthetic chimeric flavivirusnucleic acid sequence 19 ctgaacggga gaaaaagatc gatcattgac ggcttgaactcgaggaacac t 51 20 17 PRT Artificial Sequence Synthetic chimericflavivirus protein sequence 20 Leu Asn Gly Arg Lys Arg Ser Ala Val AspGly Leu Asn Ser Arg Asn 1 5 10 15 Thr 21 51 DNA Artificial SequenceSynthetic chimeric flavivirus nucleic acid sequence 21 ctgaacgggagaaaaaggtc tgcagttgac ggcttgaact cgaggaacac t 51 22 17 PRT ArtificialSequence Synthetic chimeric flavivirus protein sequence 22 Met Ala PheSer Leu Val Ala Arg Glu Arg Gly Leu Asn Ser Arg Asn 1 5 10 15 Thr 23 51DNA Artificial Sequence Synthetic chimeric flavivirus nucleic acidsequence 23 atggcgtttt ccttggttgc aagagagaga ggcttgaact cgaggaacac t 5124 17 PRT Artificial Sequence Synthetic chimeric flavivirus proteinsequence 24 Gly Thr Asn Ser Arg Asn Pro Thr Arg Gly Arg Arg Ser Trp ProLeu 1 5 10 15 Asn 25 51 DNA Artificial Sequence Synthetic chimericflavivirus nucleic acid sequence 25 ggcacgaact cgaggaaccc aaccagggggagacgatcgt ggcctcttaa c 51 26 17 PRT Artificial Sequence Syntheticchimeric flavivirus protein sequence 26 Gly Thr Asn Ser Arg Asn Pro ThrGly Thr Gly Trp Ile Arg Lys Lys 1 5 10 15 Arg 27 51 DNA ArtificialSequence Synthetic chimeric flavivirus nucleic acid sequence 27ggcacgaact cgaggaaccc aaccggcacg ggctggattc gaaagaaaag a 51 28 17 PRTArtificial Sequence Synthetic chimeric flavivirus protein sequence 28Gly Ala Ser Arg Arg Ser Phe Asn Glu Ser Gly Arg Arg Ser Ile Thr 1 5 1015 Leu 29 51 DNA Artificial Sequence Synthetic chimeric flavivirusnucleic acid sequence 29 ggagcctcaa gacgatcgtt taatgagtct ggaagaagatctataactct c 51

What is claimed is:
 1. A viable chimeric recombinant flavivirus,comprising: a first region of nucleic acid operably encoding preM and Estructural proteins of a Langat virus operably linked to a second regionof nucleic acid operably encoding non-structural proteins of a dengue 1virus, dengue 2 virus, dengue 3 virus, or dengue 4 virus, wherein saidLangat virus is defined as an attenuated tick-borne encephalitis virus.2. The recombinant flavivirus of claim 1, wherein said Langat virus isLangat strain TP21 or Langat strain B5.
 3. The recombinant flavivirus ofclaim 1, wherein said first region of nucleic acid also operably encodescapsid protein from said dengue 1 virus, dengue 2 virus, dengue 3 virus,or dengue 4 virus.
 4. The recombinant flavivirus of claim 1, furthercomprising at least one mutation that does not cause neurovirulence orneuroinvasiveness.
 5. An immunogenic composition for generating animmune response against tick-borne encephalitis virus or one of itshighly virulent tick-borne flavivirus relatives comprising the chimericviable recombinant flavivirus of claim 1 in a pharmaceuticallyacceptable carrier.
 6. A recombinant DNA construct comprising: a firstregion of nucleic acid operably encoding preM and E structural proteinsof a Langat virus operably linked to a second region of nucleic acidoperably encoding non-structural proteins of a dengue 1 virus, dengue 2virus, dengue 3 virus, or dengue 4 virus incorporated within anexpression vector, wherein said Langat virus is defined as an attenuatedtick-borne encephalitis virus.
 7. The recombinant DNA construct of claim6, wherein said vector is a plasmid.
 8. A host cell stably transformedwith the recombinant DNA construct of claim 5, in a manner allowingexpression of said DNA construct.
 9. The host cell of claim 8, whereinsaid host cell is a prokaryotic cell.
 10. The immunogenic composition ofclaim 5, wherein said tick-borne encephalitis virus or one of its highlyvirulent tick-borne flavivirus relatives is the Eastern or Westernsubtype or Omsk hemorrhagic fever, louping ill, Kyasanur forest disease,Negishi, or Powassan virus.